Trypsin active site

The reaction process of trypsin-catalyzed hydrolysis of the inverse substrates is illustrated in Fig. 4. Here the process is compared to that of normal-type substrates. After specific binding and efficient acylation, the site-specific amidinophenyl moiety is cleaved (leaving group) to give the acyl enzyme in a very specific manner. As a result, inverse substrates are expected to be applicable as a general method for specific introduction of any acyl group of non-specific structure into the trypsin active site. 

For the first time, inverse substrates provide a general method for the specific introduction of an acyl group into the trypsin active site without recourse to cation-containing acyl compounds. The preparation of various new acyl enzymes is expected to lead to the discovery of novel features of the enzymatic reaction mechanism. In addition, any desired reporter groups might be specifically introduced into the trypsin... 

The microenvironment of the trypsin active site was estimated by spectrometric analysis of these acyl trypsin preparations. 

The third criterion of the inhibition of fertilization by active site inhibitors was observed both In vitro (63) and in vivo (64) fertilization. However, this inhibition was never total, and once the sperm had tightly bound the ZP, trypsin active site inhibitors did not prevent further penetration or hydrolysis of the ZP (65). Therefore, while acrosin does not strictly follow all of the criteria for a ZP lysin, it does cause limited hydrolysis of the ZP and it is in the correct place to act in penetration of the ZP. 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

Fig. I. An electrostatic potential molecular surface for the trypsin active site and for trypsin inhibitor [25].

Fig. 10.12 Sequence alignment of trypsin, chymotrypsin and thrombin (bovine). The active sites histidine, aspartic acid and serine are highlighted.

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

FIGURE 5.9. The relationship between Ag and AG for proton transfer reactions in the active site of trypsin. 

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

Feldmann, RJ, Bing DH, Furie BC, Furie B. Interactive computer surface graphics approach to study of the active site of bovine trypsin. Proc Natl Acad Sci USA 1978 75 5409-12. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

The outstanding inclusion ability and the carboxylic functions of host I raised the idea of co-erystallizing it with imidazole (Im) which, due to its versatile nature 114), is one of the frequently used components in enzyme active sites, generally presented by histidine. Formally, a system made of imidazole and an acid component may mimic two essential components of the so-called catalytic triad of the serine protease family of enzymes the acid function of Aspl02 and the imidazole nucleus of His57 115) (trypsin sequence numbering). The third (albeit essential) component of the triad corresponding to the alcohol function of Seri 95 was not considered in this attempt. This family of enzymes is of prime importance in metabolitic processes. 

Trypsinogen, the inactive proenzyme form of trypsin has no water molecules in its unordered active site cf. Fehlhammer, H., Bode, W., Huber, R. ibid. Ill, 415 (1977)... 

Thus the alkaline protease obtained from Bacillus licheniformis with a molecular mass of about 27 000 consists of 274 amino acid residues and has serine and histidine as active sites. Pancreatic trypsin with a molecular mass of about 24 000 contains 230 amino acid residues and also has serine and histidine as active sites. Papain (molecular mass about 23 000 and 211 amino acid residues) has cysteine and histidine as active sites. 

Boldicke, T., Kindt, S., Maywald, F., Fitzlaff, G., Bocher, M., Frank, R., and Collins, J. (1988) Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides. Eur. J. Biochem. 175, 259-264. 

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